Friedreich's ataxia (FRDA) is a debilitating cardio- and neurodegenerative disease currently affecting 6,000 Americans. Recent progress in FRDA research has linked pathogenesis of FRDA to the absence of Frataxin (Fxn), a protein that regulates iron-sulfur cluster (Fe-S) biosynthesis, possibly by serving as a regulator of the pathway or as an iron chaperone that delivers metal to protein partners in the Fe-S cluster bioassembly (ISC) pathway. In eukaryotes, the predominant ISC assembly pathway is located within the mitochondria and driven by coordinated association of the bioassembly scaffold protein (Isu1), a cysteine desulfurase that provides atomic sulfur (Nfs1), an accessory protein (Isd11) and frataxin (Yfh1). The long-range goal of the Stemmler laboratory is to characterize the molecular details of the Fe-S cluster assembly pathway in eukaryotes. The objective of this proposal is to characterize the active site of the Isu1 scaffolding protein in addition to the overll ISC assembly complex. The findings of this study will help in the development strategies to augment the activity of this pathway that is deficient when frataxin is absent. All studies will be performed in the yeast system, where the genetics of Fe-S cluster assembly have been well established. Our preliminary hypothesis is that Fxn provides two Fe (II) atoms and Nfs provides two sulfur atoms at distinct sites on Isu1. Subsequent Isu1 rearrangement brings both sulfur and Fe (II) species together at Isu1's active site to accomplish cluster assembly. This proposal follows two specific aims. Firstly, Isu1 will be manipulated to identify and characterize critical residues essential for Fe-S assembly. Secondly, a multi-protein expression plasmid, with all 4 ISC complex proteins present, will be utilized to determine how Isu1, Nfs1, Isd11 assemble in the presence/absence of frataxin. As a whole, this proposal will facilitate the development of novel therapeutic strategies for FRDA.